The aim of drug targeting is to kill target cells such as cancer cells, while leaving normal tissues unharmed. Immunotoxins combine the selectivity of an antibody moiety with the potency of a toxin moiety. Such agents kill target cells via a process which involves specific binding to a cell surface antigen preferentially expressed on the cells, such as a tumor-associated antigen, internalization and delivery of the toxic moiety to the cytosol, where a critical cell function is inhibited, leading to cell death. A decisive breakthrough in immunotoxin development was the advent of hybridoma technology, making monoclonal antibodies (mAbs) available in limitless supply. The “first generation” of immunotoxins, for example as disclosed in U.S. Pat. No. 4,545,985, were chemically linked conjugates of mAbs or Fab′ fragments capable of binding cancer cell antigens, and potent protein toxins derived from plants or bacteria such as ricin, abrin, saporin, Pseudomonas aeruginosa exotoxin (PE), cholera toxin (CT) and Diphtheria toxin (DT). Such early immunotoxins showed impressive results in vitro but in most cases displayed poor anti-tumor effects in animals or humans, and often with excessive toxicity.
The “second generation” of immunotoxins were generally fully recombinant antibody-toxin chimeric molecules, usually in the form of a single-chain antibody genetically fused to a truncated version of either DT or PE, such as disclosed for example in U.S. Pat. No. 6,051,405.
Over the years, a large number of antibodies that bind tumor-associated antigens have been isolated. Early on, the need for a rapid screening approach to assess the potential of such antibodies was recognized, since internalization is a pre-requisite for most drug delivery approaches (Casalini et al 1993, Cancer Immunol Immunother 37, 54-60). An undisputed proof of internalization can be provided by linking the antibody to a cytotoxic cargo (such as a drug or a toxin) and testing the ability of the antibody to deliver the cargo into a target cell. The first generation of immunotoxins could provide such a tool, but some antibodies are not readily conjugated. While use of second generation recombinant immunotoxins for screening purposes is technically feasible, it is extremely labor intensive.
Some agents that could potentially link any IgG to a toxin have been disclosed, for example in Kim and Weaver 1998, Gene 68, 315-21; O'Hare et al 1990, FEBS Lett 273, 200-4; Madshus et al 1991, J Biol Chem 266, 17446-53; Tonevitskii et al 1991, Mol Biol (Mosk) 25, 1188-96, but none of these disclosures show an agent effective in target cell killing.
The immunoglobulin Fc-binding protein denoted ZZ is a recombinant tandem repeated, mutated form of domain B of protein A from Staphylococcus aureus which has been used in a variety of biotechnological applications (Nilsson et al 1987, Protein Eng. 1, 107-13; Nilsson et al 1996, Protein Eng. 1, 107-13).
Fusion proteins composed of protein ZZ and diphtheria toxin, either the full-length toxin or fragment B thereof, have been disclosed (Madshus et al 1991, supra; Nizard et al 1998, FEBS Lett 433, 83-8). A chimeric protein composed of S. aureus protein A fragments and Pseudomonas aeruginosa exotoxin A has been disclosed (Tonevitskii et al 1991, supra). While the chimeric protein was shown to be capable of ADP-ribosylation of elongation factor 2 and binding to immunoglobulin, evaluation of its cytotoxic properties in two model systems showed only a slight inhibition of target cell growth.
U.S. Pat. No. 5,917,026 discloses DNA sequences encoding fusion proteins comprising a first segment which encodes a native or mutant subunit of a bacterial toxin that confers enzymatic ADP-ribosylating activity inter alia Pseudomonas toxin, and a second segment which encodes a peptide which confers water solubility on the fusion protein and targets the fusion protein to a specific cell receptor different from receptors binding to the native toxin, and can thereby mediate intracellular uptake of at least the toxin subunit. According to the disclosure, the receptor may be one present on B lymphocytes and the peptide may be inter alia S. aureus protein A or a fragment thereof in single or multiple copies. The only fusion proteins specifically disclosed are those composed of cholera toxin subunit A1 linked to DD, the latter being a dimer of the D-region of protein A, and such fusions are described as being non-toxic in vivo and capable of enhancing immune effects of B and T cells. According to the disclosure, the intended use of the fusion protein is for potentiating immune responses.
There remains a need for effective immunotoxins and recombinant reagents for screening of antibodies for their potential as components of such immunotoxins, in particular the ability to be internalized within target cells.